U.S. patent application number 15/322274 was filed with the patent office on 2017-05-18 for compositions and methods for treating diabetes.
The applicant listed for this patent is The Children's Medical Center Corporation. Invention is credited to Hilde Herrema, Umut Ozcan.
Application Number | 20170137800 15/322274 |
Document ID | / |
Family ID | 53674356 |
Filed Date | 2017-05-18 |
United States Patent
Application |
20170137800 |
Kind Code |
A1 |
Ozcan; Umut ; et
al. |
May 18, 2017 |
COMPOSITIONS AND METHODS FOR TREATING DIABETES
Abstract
Compositions containing an FKBP 11 peptide (i.e., FKBP11
polypeptide, a variant or a fragment thereof), a fusion protein
containing an FKBP11 peptide, or a nucleic acid encoding an FKBP 11
peptide are disclosed. Also disclosed are methods of reducing blood
glucose levels, improving glucose tolerance, decreasing hepatic
gluconeogenic activity and/or improving insulin sensitivity in a
subject, by administering a composition containing an FKBP 11
peptide or a fusion protein containing an FKBP11 peptide. The
methods can include administering nucleic acids encoding an FKBP 11
peptide or a fusion protein containing an FK BP11 peptide, to the
subject, or cells expressing the nucleic acids. Kits containing an
FKBP 11 peptide, are also provided.
Inventors: |
Ozcan; Umut; (Boston,
MA) ; Herrema; Hilde; (Amsterdam, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Children's Medical Center Corporation |
Boston |
MA |
US |
|
|
Family ID: |
53674356 |
Appl. No.: |
15/322274 |
Filed: |
July 8, 2015 |
PCT Filed: |
July 8, 2015 |
PCT NO: |
PCT/US2015/039576 |
371 Date: |
December 27, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62021859 |
Jul 8, 2014 |
|
|
|
62087566 |
Dec 4, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/40 20130101;
A61K 38/52 20130101; C12N 9/90 20130101; A61K 38/00 20130101; A61K
48/005 20130101; C12Y 502/01008 20130101; C07K 14/4702 20130101;
A61P 3/10 20180101 |
International
Class: |
C12N 9/90 20060101
C12N009/90 |
Claims
1. A composition for increasing the levels of an FKBP11 peptide in
a subject comprising an FKBP11 peptide or a nucleic acid encoding
an FKBP11 peptide, wherein the FKBP11 peptide is selected from the
group consisting of an FKBP11 polypeptide, a variant or a fragment
thereof or a fusion protein containing an FKBP11 polypeptide, a
variant or a fragment thereof.
2. The composition of claim 1, in a form selected from the group
consisting of dry powders, tablets, wafers, films, lozenges, and
capsules.
3. The composition of claim 1, in a form suitable for parenteral
administration.
4. The composition of claim 1 wherein the FKBP11 polypeptide
comprises SEQ ID NO: 1.
5. The composition of claim 1 comprising a nucleic acid encoding an
FKBP11 polypeptide, a variant or a fragment thereof in a
vector.
6. The composition of claim 5, wherein the vector is selected from
the group consisting of bacteriophage, baculoviruses, tobacco
mosaic virus, herpes viruses, cytomegalo virus, retroviruses,
vaccinia viruses, adenoviruses, and adeno-associated viruses.
7. The composition of claim 1, wherein the FKBP11 peptide reduces
blood glucose levels in the subject.
8. A method of increasing the levels of FKBP11 peptide in a subject
comprising administering a composition comprising an FKBP11
peptide, or a nucleic acid encoding an peptide, wherein the FKBP11
peptide is selected from the group consisting of an FKBP11
polypeptide, a variant or a fragment thereof or a fusion protein
containing an FKBP11 polypeptide, a variant or a fragment
thereof.
9. The method of claim 8, wherein the wherein the subject is
selected from a group consisting of a subject with type I diabetes,
a subject with type II diabetes, an obese subject, a gestational
diabetic, and a subject exhibiting insulin tolerance
10. The method of claim 8, wherein the FKBP11 peptide reduces blood
glucose levels in the subject.
11. The method of claim 8, wherein the composition is in a form
selected from the group consisting of dry powders, tablets, wafers,
films, lozenges, and capsules.
12. The method of claim 8, wherein the composition is administered
parenterally.
13. The method of claim 8, further comprising transforming one or
more cells ex vivo to express the nucleic acid encoding an FKBP11
peptide and transferring the transformed cells into the
subject.
14. The method of claim 13, wherein the cells are selected from the
group consisting of pancreatic cells, islet cells and pancreatic
precursor cells.
15. A kit comprising a first storage container and a second storage
container, wherein the first storage contain comprises an FKBP11
peptide, or a nucleic acid encoding an FKBP11 peptide, and wherein
the second storage container comprises an excipient, wherein the
FKBP11 peptide is selected from the group consisting of an FKBP11
polypeptide, a variant or a fragment thereof or a fusion protein
containing an FKBP11 polypeptide, a variant or a fragment
thereof.
16. The kit of claim 15 wherein the first container is a cap and
the second container is a vial the cap is secured to, and wherein
the two containers are separated by a barrier.
Description
FIELD OF THE INVENTION
[0001] The invention is generally related to the field of metabolic
homeostasis, more particularly to methods and compositions for
lowering blood glucose levels, and treating diabetes.
BACKGROUND OF THE INVENTION
[0002] Diabetes mellitus (DM) is a group of metabolic diseases
where the subject has high blood sugar, either because the pancreas
does not produce enough insulin, or, because cells do not respond
to insulin that is produced. Diabetes affects more than 25.8
million people in the United States alone, i.e.
[0003] 8.3% of the population. About 1 9 million people aged 20
years or older were newly diagnosed with diabetes in 2010. An
estimated 79 million people aged 20 years or older are believed to
have prediabetes, which constitutes 5% of adults aged 20 years or
older and 50% of adults aged 65 years or older. National Diabetes
Information Clearinghouse, National Diabetes Statistics, 2011.
[0004] Much of the morbidity and cost of diabetes management is
attributable to long-term diabetes-related complications. For
example, diabetes is the leading cause of kidney failure,
non-traumatic lower limb amputations and new cases of blindness
among adults. Diabetes is also a major cause of heart disease and
stroke. After adjusting for population age and sex differences,
average medical expenditures among people with diagnosed diabetes
were 2.3 times higher than the expected expenditures without
diabetes. The chronic elevation of blood glucose level associated
with DM leads to damage of blood vessels. The resulting problems
are grouped under "microvascular disease" (due to damage to small
blood vessels) and "macrovascular disease" (due to damage to the
arteries). The damage to small blood vessels leads to a
microangiopathy, which can cause diabetic retinopathy and/or
diabetic nephropathy. Microvascular complications including
retinopathy and nephropathy account for the most prevalent and
severe morbidity associated with diabetes and may be involved in
mediating the increased risk of cardio- and cerebrovascular disease
as well. Diabetes is also the leading cause of renal insufficiency
and end-stage renal disease (ESRD) in the U.S., and the Western
world. Although diabetic microvascular complications are clearly
associated with the degree of hyperglycemia, not all diabetic
individuals with poor glycemic control develop renal or advanced
retinal complications. Conversely, some diabetic patients develop
severe complications despite well-controlled blood glucose
concentrations.
[0005] There are two main types of diabetes. Type 1 diabetes
results from the body's failure to produce insulin. Type 2 diabetes
results from insulin resistance, a condition in which cells fail to
use insulin properly, sometimes combined with an absolute insulin
deficiency. This form was previously referred to as non
insulin-dependent diabetes mellitus (NIDDM) or "adult-onset
diabetes". A third form, gestational diabetes occurs when pregnant
women without a previous diagnosis of diabetes develop a high blood
glucose level. It may precede development of type 2 diabetes, or it
may resolve at the end of the pregnancy.
[0006] The cost of diabetes in 2007 was $175 billion, which
includes $116 billion in excess medical expenditures and $58
billion in reduced national productivity. Dall, et al., Diabetes
Care, 31(3):596-615 (2008). Because patients with Type 1 diabetes
produce no insulin, the primary treatment for Type 1 diabetes is
daily intensive insulin therapy. The treatment of Type 2 diabetes
typically starts with management of diet and exercise. Although
helpful in the short-run, treatment through diet and exercise alone
is not an effective long-term solution for the vast majority of
patients with Type 2 diabetes. When diet and exercise are no longer
sufficient, treatment commences with various non-insulin oral
medications. These oral medications act by increasing the amount of
insulin produced by the pancreas, by increasing the sensitivity of
insulin-sensitive cells, by reducing the glucose output of the
liver or by some combination of these mechanisms. These treatments
are limited in their ability to manage the disease effectively and
generally have significant side effects, such as weight gain and
hypertension. Because of the limitations of non-insulin treatments,
many patients with Type 2 diabetes progress over time and
eventually require insulin therapy to support their metabolism.
Many of the known hypoglycemic agents exhibit undesirable side
effects and are toxic in certain cases. Accordingly, there is a
need for additional methods and compositions for treating
diabetes.
[0007] It is an object of the present invention to compositions for
treating diabetes in a subject.
[0008] It is also an object of the present of the present invention
to provide a method for treating diabetes in a subject.
[0009] It is a further object of the invention to provide kits for
treating diabetes in a subject.
SUMMARY OF THE INVENTION
[0010] The compositions provided herein are based on the discovery
that FK506-binding protein 11 (FKBP 11) plays a role in glucose
metabolism. FKBP 11 lowers blood glucose levels, improve glucose
tolerance, decreases hepatic gluconeogenic activity and/or insulin
sensitivity in a subject.
[0011] Compositions containing an effective amount of FKBP 11
peptide can be used to treat a subject diagnosed with type 1 or
type 2 diabetes to lower blood glucose levels, improve glucose
tolerance, decrease hepatic gluconeogenic activity and/or insulin
sensitivity in a subject. The compositions disclosed herein can
include nucleic acids encoding FKBP 11 peptide or a fusion protein
including an FKBP 11 peptide, vectors containing such nucleic acids
and host cells expressing the vectors, either for administration of
the nucleic acid to an individual or for expression of protein for
administration to an individual. In one embodiment, the host cell
is a mammalian cell, preferably a human cell, more preferably, a
pancreatic cell or pancreatic progenitor cell. In still other
embodiments, the host cell is a yeast cell. In other embodiments
the cell is a prokacryotic cell. The host cell may also be used in
a screening assay for agents which upregulate/down regulate glucose
modulating activities of an FKBP 11 peptide.
[0012] Also provided is a method of controlling blood glucose
levels, improve glucose tolerance, decrease hepatic gluconeogenic
activity and/or insulin sensitivity in a subject, by administering
a composition containing an FKBP 11 peptide or a fusion protein
including an FKBP 11 peptide. The methods can include administering
nucleic acids encoding an FKBP 11 peptide, to the subject. In one
embodiment, the nucleic acid is administered in vivo. In another
embodiment, the nucleic acid is administered ex vivo, whereby cells
are removed from a subject, and a nucleic acid encoding an FKBP 11
peptide, or a fusion protein including an FKBP 11 peptide, is
introduced into the cells, which are then reintroduced into the
subject. The subject is preferably a mammal, more preferably, a
human subject or an animal subject, for example, domestic animals
and pets. The subject can be a type 1 diabetic, a type II diabetic,
an obese subject, a subject exhibiting higher than normal blood
glucose levels, or a gestational diabetic.
[0013] Also provided are kits containing an FKBP 11 peptide or a
fusion protein including an FKBP 11 peptide, for treating or
alleviating one or more symptoms of diabetes in a subject. The FKBP
11 peptide can be stored in one container and the excipients can be
stored in a second container. Immediately prior to administration
the contents of both containers are mixed. In one embodiment, the
kit may contain a vial containing lyophilized FKBP 11 peptide or a
fusion protein including the FKBP 11 peptide, in the cap, separated
by a seal which can be broken by rotation of the cap, to allow the
insulin to mix with the excipient solution in the vial.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1A shows gene expression levels in livers of
genetically obese and diabetic ob/ob mice compared to lean mice.
FIG. 1B shows gene expression levels in high fat diet (HFD)-induced
obese and insulin resistant mice, compared to lean mice.
[0015] FIG. 2A shows gene expression following overexpression of
FKBP11 in livers of lean mice. FIGS. 2B-2D show body weight (FIG.
2B), food intake (FIG. 2C) and blood glucose levels (FIG. 2D) in
lean mice injected with FKBP11-containing adenovirus, when compared
to lean mice injected with adLacZ (control).
[0016] FIG. 3A shows gene expression following overexpression of
FKBP11 in livers of genetically obese and diabetic ob/ob mice.
FIGS. 3B-3D show the effect of overexpression of FKBP11 in livers
of ob/ob mice, on body weight (FIG. 3B), food intake (FIG. 3C), and
blood glucose levels (FIG. 3D).
[0017] FIG. 4A shows the effect of overexpression of FKBP11 on
glucose tolerance as assessed by glucose tolerance testing (GTT) in
lean mice (area under the curve for FIG. 4A is depicted in FIG.
4B). FIG. 4C shows the effect of overexpression of FKBP11 on
insulin tolerance as assessed by means of an insulin tolerance test
(ITT). FIG. 4D shows the effect of overexpression of FKBP11 on
glucose tolerance in ob/ob mice as assessed by GTT (area under the
curve for FIG. 4D is depicted in FIG. 4E). FIG. 4F shows the effect
of overexpression of FKBP11 on insulin tolerance as assessed by
means of an insulin tolerance test (ITT).
[0018] FIGS. 5A-5D show the effect of FKPB11 overexpression on
hepatic glucose production as assessed by pyruvate tolerance test
(PTT) in lean and obese mice. FIGS. 5A and 5B show glucose levels
and the AUC for PTT, respectively, for lean mice. FIGS. 5C and 5D
show glucose levels as assessed by pyruvate tolerance test (PTT)
and AUC for PTT respectively, in ob/ob mice.
[0019] FIG. 6A shows gene expression levels for FKBP11 in HFD-fed
mice that overexpress FKBP11. FIGS. 6B-6E show the effect of FKBP11
on food intake (FIG. 6B), body weight (FIG. 6C) and glucose levels
(FIGS. 6D and 6E). AUC for FIG. 6E is depicted in FIG. 6F.
[0020] FIG. 7A shows endogenous gene expression levels of FKBP11 in
livers of STZ-induced type I diabetic mice. FIGS. 7B and show
hepatic gene and protein expression of STZ-induced type I diabetic
mice that overexpress FKBP11. FIGS. 7C to 7F show the effect of
FKBP11 on insulin levels (FIG. 7C), body weight (FIG. 7D), food
intake (FIG. 7E), and blood glucose levels (FIG. 7F).
[0021] FIG. 8 shows FKBP11 ELISA read outs (A450 nm) from cell
culture media of HEK cells overexpressing FKBP11 are presented.
[0022] FIG. 9 shows blood glucose levels following iv
administration of recombinant FKBP11 in diabetic mice, when
compared to control (Buffer).
DETAILED DESCRIPTION OF THE INVENTION
[0023] It has been discovered that there is a direct link between
low levels of secreted FKBP11 and glucose metabolism. As
demonstrated by the examples, FKBP11 is involved in maintaining
glucose homeostasis in obese and type 2 diabetic mice, as well as
in a mouse model of type 1 diabetes. FKBP11 expression is
dynamically regulated in healthy lean mice that are subjected to
metabolic stress such as refeeding after a fasting period,
indicating an important physiological role in metabolic control.
Hepatic expression levels of FKBP11 are reduced in obese and type 2
diabetic mice.
[0024] Restoring FKBP11 levels dramatically reduced fed and fasted
blood glucose levels, and improved glucose tolerance, hepatic
gluconeogenic activity and insulin sensitivity. FKBP11 expression
also reduced glucose levels in a mouse model of type-I
diabetes.
[0025] Accordingly, compositions and methods for reducing glucose
levels, improving glucose tolerance and improving insulin
sensitivity, by increasing FKBP11 peptide in a subject, are
provided. The preferred FKBP11 is an FKBP11 polypeptide,
represented by SEQ ID NO: 1.
I. Definitions
[0026] "Effective amount" is used herein to refer to a sufficient
amount of an agent to provide a desired effect. The exact amount
required will vary from subject to subject, depending on the
species, age, and general condition of the subject, the severity of
disease that is being treated, the particular agent used, and its
mode of administration. An appropriate "effective amount" may be
determined empirically by one of ordinary skill in the art using
routine methods.
[0027] "Expression vector" is used herein to refer to a vector that
includes one or more expression control sequences.
[0028] "Expression control sequence" is used herein to refer to a
DNA sequence that controls and regulates the transcription and/or
translation of another DNA sequence.
[0029] "FKBP11" is used herein interchangeably with "FKBP19". It
belongs to a family of proteins known as peptidyl-prolyl cis/trans
isomerases (PPIase) involved in folding of proline-containing
polypeptides.
[0030] "FKBP11 polypeptide, fragments thereof, variants thereof are
collectively referred to herein as "FKBP11 peptides".
[0031] "Identity," as known in the art, is a relationship between
two or more polypeptide sequences, as determined by comparing the
sequences. In the art, "Identity" also means the degree of sequence
relatedness between polypeptide as determined by the match between
strings of such sequences.
[0032] "Insulin resistance" is used herein to refer to a
physiological condition in a subject where insulin becomes less
effective at lowering blood sugars (low insulin sensitivity), which
results in an increase in blood glucose. Insulin resistance in
muscle and fat cells reduces glucose uptake, whereas insulin
resistance in liver cells results in reduced glycogen synthesis and
storage and a failure to suppress glucose production and release
into the blood.
[0033] "Isolated nucleic acid" is used herein to refer to a nucleic
acid that is separated from other nucleic acid molecules that are
present in a mammalian genome, including nucleic acids that
normally flank one or both sides of the nucleic acid in a mammalian
genome. The tenn "isolated" as used herein with respect to nucleic
acids also includes the combination with any
non-naturally-occurring nucleic acid sequence, since such
non-naturally-occurring sequences are not found in nature and do
not have immediately contiguous sequences in a naturally-occurring
genome.
[0034] "Low stringency" as used herein refers to conditions that
peimit a polynucleotide or polypeptide to bind to another substance
with little or no sequence specificity.
[0035] "Pharmaceutically acceptable carrier" as used herein
encompasses any of the standard pharmaceutical carriers, such as a
phosphate buffered saline solution, water and emulsions such as an
oil/water or water/oil emulsion, and various types of wetting
agents.
[0036] "Protein transduction domain" or "PTD" refers to a
polypeptide, polynucleotide, carbohydrate, organic or inorganic
compound that facilitates traversing a lipid bilayer, micelle, cell
membrane, organelle membrane, or vesicle membrane.
[0037] "Purified" and similar terms as used herein relate to the
isolation of a molecule or compound in a form that is substantially
free (at least 60% free, preferably 75% free, and most preferably
90% free) from other components normally associated with the
molecule or compound in a native environment.
[0038] The term "treatment" refers to the medical management of a
subject with the intent to cure, ameliorate, stabilize, or prevent
one more symptoms of a disease, pathological condition, or
disorder. This term includes active treatment, that is, treatment
directed specifically toward the improvement of a disease,
pathological condition, or disorder, and also includes causal
treatment, that is, treatment directed toward removal of the cause
of the associated disease, pathological condition, or disorder. In
addition, this term includes palliative treatment, that is,
treatment designed for the relief of symptoms rather than the
curing of the disease, pathological condition, or disorder;
preventative treatment, that is, treatment directed to minimizing
or partially or completely inhibiting the development of the
associated disease, pathological condition, or disorder; and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder.
[0039] "Transformed" and "transfected" are used herein to encompass
the introduction of a nucleic acid (e.g. a vector) into a cell by a
number of techniques known in the art.
[0040] "Variant" refers to a polypeptide or polynucleotide that
differs from a reference polypeptide or polynucleotide, but retains
essential properties.
[0041] "Vector" as used herein refers to a replicon, such as a
plasmid, phage, virus or cosmid, into which another DNA segment may
be inserted so as to bring about the replication of the inserted
segment. Vectors can be expression vectors.
II. Compositions
[0042] Compositions for increasing FKPB11 polypeptide include
formulations containing a purified FKPB11 peptide. Compositions for
increasing FKPB11 polypeptide also include vectors containing
nucleic acid sequences encoding an FKBP11 peptide. FKBP11 peptides
include FKBP11 polypeptide, fragments thereof, variants thereof and
fusion peptides containing an FKBP11 peptide.
[0043] Purified FKBP11 peptides can be obtained by expressing and
amplifying a vector containing a tagged (e.g., 6*HIS) form of
FKBP11 in eukaryotic cells (preferred), insect cells or bacteria.
Tagged FKBP11 will be expressed the cells and can subsequently be
purified from cell lysate or cell culture media by
antibody-mediated pull down (the antibody recognizes the tag, which
allows for clean and efficient isolation of FKBP11). Since some
tags interfere with protein activity/specificity, it is possible to
have the tag removed after the isolation and purification
process.
[0044] Formulations containing an isolated FKBP11 peptide as an
active agent also contain one or more pharmaceutically suitable
excipients. FKBP11 peptides may be administered in the form of a
pharmaceutical composition wherein the FKBP11 is in admixture or
mixture with one or more pharmaceutically acceptable carriers,
excipients or diluents.
[0045] In some embodiments, the FKPB11 peptide may be administered
as a pharmaceutically acceptable acid- or base-addition salt,
formed by reaction with inorganic acids such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases such as mono-, di-, trialkyl and aryl
amines and substituted ethanolamines.
[0046] A. FKBP11 Peptides-FKBP11 Polypeptides, Fragments/Variants
Thereof, and Fusion Proteins Containing FKBP11
[0047] FKBP11 belongs to a family of proteins known as
peptidyl-prolyl cis/trans isomerases (PPIase) involved in folding
of proline-containing polypeptides. The PPIase families are
classified by sequence homology and pharmacologically by their
ability to bind the immunosuppressant compounds cyclosporine, FK506
and rapamycin, and are otherwise known as immunophilins. The
FK506-binding protein (FKBP) family shares a high degree of
sequence and structural homology and PPIase activity that is
specifically inhibited by FK506 or rapamycin. Since the discovery
of the first FKBP several members of this family have been
characterized in humans and other organisms (Reviewed in Sulten, et
al., in Mamm. Genome, 17(4):322-331 (2006).
[0048] 1. FKB11 Polypeptide The human FKBP11 sequence is known
(AF238079_1) mtlrpsllpl hlllllllsa avcraeagle tespvrtlqv etiveppepc
aepaafgdtl hihytgslvd griidtsltr dplvielgqk qvipgleqsl ldmcvgekrr
aiipshlayg krgfppsvpa davvqydvel ialiranywl klvkgilplv gmamvpallg
ligyhlyrka nrpkvskkkl keekrnkskk k (SEQ ID NO: 1)
[0049] FKBP19 includes a leucine-rich N-terminal leader sequence of
25 residues, which shows similarities with other known secretory
pathway proteins. Cleavage at the predicted site of 3 kDa leaves a
19 kDa mature protein, thus named FKBP19. Anti-FKBP19 was used to
detect a doublet of 19-22 kDa in bovine pancreas extracts
Immunohistochemical analysis of FKBP19 production in the mouse
pancreas shows high levels of FKBP19 protein, localized throughout
the cytoplasmic region of acinar cells and concentrated in the
perinuclear region of these cells. Low levels are seen in the
islets of Langerhans. (Sulten, et al., in Mamm. Genome,
17(4):322-331 (2006)).
[0050] FKBP11 has high (around 90%) sequence homology in mice,
humans and rats. There are 3 isoforms of FKBP11 predicted in humans
The first domain is a signal peptide, which targets FKBP11 to the
secretory pathway. This domain is predicted to be cleaved after AA
25. The second domain is predicted to be a peptidylprolyl isomerase
(PPIase) domain, which potentially serves as enzymatic domain. The
PPIase domain is highly conserved amongst the FKBP protein family
members and for some, but not all, of the FKBP family members,
their function is determined by PPIase activity. The third domain
is a hydrophobic domain that is predicted to be a transmembrane
domain. Similar hydrophobic sequences are found in type I
transmembrane protein family of proteins and accordingly, FKBP11
can be categorized as such. Some of these proteins are known to
have a cleavage site near the hydrophobic domain that following
cleavage release a soluble fragment leaving the transmembrane
domain residing in the membrane.
[0051] 2. Variants/Fragments of the FKBP11 Polypeptide
[0052] A typical variant of a polypeptide differs in amino acid
sequence from another, reference polypeptide. Generally,
differences are limited so that the sequences of the reference
polypeptide and the variant are closely similar overall and, in
many regions, identical. A variant and reference polypeptide may
differ in amino acid sequence by one or more modifications (e.g.,
substitutions, additions, and/or deletions). A substituted or
inserted amino acid residue may or may not be one encoded by the
genetic code. A variant of a polypeptide may be naturally occurring
such as an allelic variant, or it may be a variant that is not
known to occur naturally.
[0053] Modifications and changes can be made in the structure of
the polypeptides disclosed herein and still obtain a molecule
having similar characteristics as the polypeptide (e.g., a
conservative amino acid substitution). For example, certain amino
acids can be substituted for other amino acids in a sequence,
without appreciable loss of activity. Since it is the interactive
capacity and nature of a polypeptide that defines that
polypeptide's biological functional activity, certain amino acid
sequence substitutions can be made in a polypeptide sequence and
nevertheless obtain a polypeptide with like properties.
[0054] In making such changes, the hydropathic index of amino acids
can be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a polypeptide
is generally understood in the art. It is known that certain amino
acids can be substituted for other amino acids having a similar
hydropathic index or score and still result in a polypeptide with
similar biological activity. Each amino acid has been assigned a
hydropathic index on the basis of its hydrophobicity and charge
characteristics. Those indices are: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine
(-3.9); and arginine (-4.5).
[0055] It is believed that the relative hydropathic character of
the amino acid determines the secondary structure of the resultant
polypeptide, which in turn defines the interaction of the
polypeptide with other molecules, such as enzymes, substrates,
receptors, antibodies, and antigens. It is known in the art that an
amino acid can be substituted by another amino acid having a
similar hydropathic index and still obtain a functionally
equivalent polypeptide. In such changes, the substitution of amino
acids whose hydropathic indices are within .+-.2 is preferred,
those within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred.
[0056] Substitution of like amino acids can also be made on the
basis of hydrophilicity, particularly when the biological
functional equivalent polypeptide or peptide thereby created is
intended for use in immunological embodiments. The following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0 .+-.1); glutamate
(+3.0 .+-.1); serine (+0.3); asparagine (+0.2); glutamnine (+0.2);
glycine (0); proline (-0.5 .+-.1); threonine (-0.4); alanine
(-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3);
valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent,
and in particular, an immunologically equivalent polypeptide. In
such changes, the substitution of amino acids whose hydrophilicity
values are within .+-.2 is preferred, those within .+-.1 are
particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[0057] Amino acid substitutions are generally based on the relative
similarity of the amino acid side-chain substituents, for example,
their hydrophobicity, hydrophilicity, charge, and size. Exemplary
substitutions that take various of the foregoing characteristics
into consideration are well known to those of skill in the art and
include (original residue: exemplary substitution): (Ala: Gly,
Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln:
Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val),
(Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr:
Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). The
polypeptides can include variants having about 50%, 60%, 70%, 80%,
90%, and 95% sequence identity to the polypeptide of interest.
[0058] "Identity" and "similarity" can be readily calculated by
known methods, such as those described in (Computational Molecular
Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and
Carillo and Lipman, SIAM J Applied Math, 48: 1073 (1988).
[0059] Preferred methods to determine identity are designed to give
the largest match between the sequences tested. Methods to
determine identity and similarity are codified in publicly
available computer programs. The percent identity between two
sequences can be determined by using analysis software (i.e.,
Sequence Analysis Software Package of the Genetics Computer Group,
Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol.
Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST). The
default parameters are used to determine the identity for the
polypeptides of the present disclosure.
[0060] By way of example, a polypeptide sequence may be identical
to the reference sequence, that is be 100% identical, or it may
include up to a certain integer number of amino acid alterations as
compared to the reference sequence such that the % identity is less
than 100%. Such alterations include at least one amino acid
deletion, substitution, including conservative and non-conservative
substitution, or insertion, wherein the alterations may occur at
the amino- or carboxy-terminal positions of the reference
polypeptide sequence or anywhere between those terminal positions,
interspersed either individually among the amino acids in the
reference sequence or in one or more contiguous groups within the
reference sequence. The number of amino acid alterations for a
given % identity is determined by multiplying the total number of
amino acids in the reference polypeptide by the numerical percent
of the respective percent identity (divided by 100) and then
subtracting that product from the total number of amino acids in
the reference polypeptide.
[0061] 3. Fusion Proteins Containing FKBP11 Peptides
[0062] Fusion proteins, also known as chimeric proteins, are
proteins created through the joining of two or more genes which
originally coded for separate proteins. Translation of this fusion
gene results in a single polypeptide with function properties
derived from each of the original proteins. Recombinant fusion
proteins can be created artificially by recombinant DNA technology
for use in biological research or therapeutics. Chimeric mutant
proteins occur naturally when a large-scale mutation, typically a
chromosomal translocation, creates a novel coding sequence
containing parts of the coding sequences from two different
genes.
[0063] The FKBP11 peptides disclosed herein can be engineered
delivered to a host as a fusion protein, which includes additional
domains such as a targeting domain
[0064] The functionality of fusion proteins is made possible by the
fact that many protein functional domains are modular. In other
words, the linear portion of a polypeptide which corresponds to a
given domain, such as a tyrosine kinase domain, may be removed from
the rest of the protein without destroying its intrinsic enzymatic
capability. Thus, any of the herein disclosed functional domains
can be used to design a fusion protein.
[0065] A recombinant fusion protein is a protein created through
genetic engineering of a fusion gene. This typically involves
removing the stop codon from a cDNA sequence coding for the first
protein, then appending the cDNA sequence of the second protein in
frame through ligation or overlap extension PCR. That DNA sequence
will then be expressed by a cell as a single protein. The protein
can be engineered to include the full sequence of both original
proteins, or only a portion of either.
[0066] If the two entities are proteins, often linker (or "spacer")
peptides are also added which make it more likely that the proteins
fold independently and behave as expected. Especially in the case
where the linkers enable protein purification, linkers in protein
or peptide fusions are sometimes engineered with cleavage sites for
proteases or chemical agents which enable the liberation of the two
separate proteins. This technique is often used for identification
and purification of proteins, by fusing a GST protein, FLAG
peptide, or a hexa-his peptide (aka: a 6.times.his-tag) which can
be isolated using nickel or cobalt resins (affinity chromatography)
Chimeric proteins can also be manufactured with toxins or
anti-bodies attached to them in order to study disease
development.
[0067] Alternatively, internal ribosome entry sites (IRES) elements
can be used to create multigene, or polycistronic, messages. IRES
elements are able to bypass the ribosome scanning model of 5'
methylated Cap dependent translation and begin translation at
internal sites (Pelletier and Sonenberg, 1988). IRES elements from
two members of the picornavirus family (polio and
encephalomyocarditis) have been described (Pelletier and Sonenberg,
1988), as well an IRES from a mammalian message (Macejak and Samow,
1991). IRES elements can be linked to heterologous open reading
frames. Multiple open reading frames can be transcribed together,
each separated by an IRES, creating polycistronic messages. By
virtue of the IRES element, each open reading frame is accessible
to ribosomes for efficient translation. Multiple genes can be
efficiently expressed using a single promoter/enhancer to
transcribe a single message (U.S. Pat. Nos. 5,925, 565 and
5,935,819; PCT/US99/05781). IRES sequences are known in the art and
include those from encephalomycarditis virus (EMCV) (Ghattas, et
al., Mol. Cell. Biol., 11:5848-5849 (1991); BiP protein (Macejak
and Sarnow, Nature, 353:91 (1991)); the Antennapedia gene of
drosophilia (exons d and e) [Oh et al., Genes & Development,
6:1643-1653 (1992)); those in polio virus [Pelletier and Sonenberg,
Nature, 334:320325 (1988); see also Mountford and Smith, TIG,
11:179-184 (1985)).
[0068] i. Protein Transduction Domain (PTD)
[0069] In some embodiments, the polynucleotide-binding polypeptide
is fusion protein modified to include a protein transduction domain
(PTD). A PTD attached to another molecule facilitates the molecule
traversing membranes, for example going from extracellular space to
intracellular space, or cytosol to within an organelle.
[0070] In preferred embodiments, the protein transduction domain is
a polypeptide. A protein transduction domain can be a polypeptide
including positively charged amino acids. Thus, some embodiments
include PTDs that are cationic or amphipathic. Protein transduction
domains (PTD), also known as a cell penetrating peptides (CPP), are
typically polypeptides including positively charged amino acids.
PTDs are known in the art, and include but are not limited to small
regions of proteins that are able to cross a cell membrane in a
receptor-independent mechanism (Kabouridis, P., Trends in
Biotechnology (11):498-503 (2003)). Although several PTDs have been
documented, the two most commonly employed PTDs are derived from
TAT (Frankel and Pabo, Cell, 55(6):1189-93(1988)) protein of HIV
and Antennapedia transcription factor from Drosophila, whose PTD is
known as Penetratin (Derossi et al., J Biol Chem., 269(14):10444-50
(1994)). Exemplary protein transduction domains include
polypeptides with 11 Arginine residues, or positively charged
polypeptides or polynucleotides having 8-15 residues, preferably
9-11 residues.
[0071] The Antennapedia homeodomain is 68 amino acid residues long
and contains four alpha helices. Penetratin is an active domain of
this protein which consists of a 16 amino acid sequence derived
from the third helix of Antennapedia. TAT protein consists of 86
amino acids and is involved in the replication of HIV-1. The TAT
PTD consists of an 11 amino acid sequence domain (residues 47 to
57; YGRKKRRQRR R (SEQ ID NO:3)) of the parent protein that appears
to be critical for uptake. Additionally, the basic domain
Tat(49-57) or RKKRRQRRR (SEQ ID NO:4) has been shown to be a PTD.
In the current literature TAT has been favored for fusion to
proteins of interest for cellular import. Several modifications to
TAT, including substitutions of Glutatmine to Alanine, i.e.,
Q.fwdarw.A, have demonstrated an increase in cellular uptake
anywhere from 90% (Wender et al., Proc Natl Acad Sci USA.,
97(24):13003-8 (2000)) to up to 33 fold in mammalian cells. (Ho et
al., Cancer Res., 61(2):474-7 (2001)).
[0072] The most efficient uptake of modified proteins was revealed
by mutagenesis experiments of TAT-PTD, showing that an 11 arginine
stretch was several orders of magnitude more efficient as an
intercellular delivery vehicle. Therefore, PTDs can include a
sequence of multiple arginine residues, referred to herein as
poly-arginine or poly-ARG. In some embodiments the sequence of
arginine residues is consecutive. In some embodiments the sequence
of arginine residues is non-consecutive. A poly-ARG can include at
least 7 arginine residues, more preferably at least 8 arginine
residues, most preferably at least 11 arginine residues. In some
embodiments, the poly-ARG includes between 7 and 15 arginine
residues, more preferably between 8 and 15 arginine residues. In
some embodiments the poly-ARG includes between 7 and 15, more
preferably between 8 and 15 consecutive arginine residues. An
example of a poly-ARG is RRRRRRR (SEQ ID NO:9). Additional
exemplary PTDs include but are not limited to;
TABLE-US-00001 (SEQ ID NO: 5) RRQRRTSKLM KR; (SEQ ID NO: 6)
GWTLNSAGYL LGKINLKALA ALAKKIL; (SEQ ID NO: 7) WEAKLAKALA KALAKHLAKA
LAKALKCEA; and (SEQ ID NO: 8) RQIKIWFQNR RMKWKK.
[0073] It is believed that following an initial ionic cell-surface
interaction, some polypeptides containing a protein transduction
domain are rapidly internalized by cells via lipid raft-dependent
macropinocytosis. For example, transduction of a TAT-fusion protein
was found to be independent of interleukin-2 receptor/raft-,
caveolar- and clathrin-mediated endocytosis and phagocytosis
(Wadia, et al., Nature Medicine, 10:310-315 (2004), and Barka, et
al., J. Histochem. Cytochem., 48(11):1453-60 (2000)). Therefore, in
some embodiments the polynucleotide-binding polypeptide includes an
endosomal escape sequence that enhances escape of the
polypeptide-binding protein from macropinosomes. The endosomal
escape sequence is part of, or consecutive with, the protein
transduction domain. In some embodiments, the endosomal escape
sequence is non-consecutive with the protein transduction domain.
In some embodiments the endosomal escape sequence includes a
portion of the hemagglutinin peptide from influenza (HA). One
example of an endosomal escape sequence includes GDIMGEWG
NEIFGAIAGF LG (SEQ ID NO:9).
[0074] In one embodiment a protein transduction domain including an
endosomal escape sequence includes the amino acid sequence
TABLE-US-00002 (SEQ ID NO: 10) RRRRRRRRRR RGEGDIMGEW GNEIFGAIAG
FLGGE.
[0075] ii. Targeting Signal or Domain
[0076] In some embodiments the polynucleotide-binding polypeptide
is modified to include one or more targeting signals or domains.
The targeting signal can include a sequence of monomers that
facilitates in vivo localization of the molecule. The monomers can
be amino acids, nucleotide or nucleoside bases, or sugar groups
such as glucose, galactose, and the like which form carbohydrate
targeting signals. Targeting signals or sequences can be specific
for a host, tissue, organ, cell, organelle, non-nuclear organelle,
or cellular compartment. For example, in some embodiments the
polynucleotide-binding polypeptide includes both a cell-specific
targeting domain and an organelle specific targeting domain to
enhance delivery of the polypeptide to a subcellular organelle of a
specific cells type.
[0077] B. Nucleic Acids Encoding FKBP11 Peptides
[0078] Nucleic acids encoding the FKBP 11 polypeptide are known in
the art ((accession number AF238079). An FKBP19 (i.e., FKBP11)
encoding nucleic acid was characterized by Sulten, et al., in Mamm.
Genome, 17(4):322-331 (2006). The 727 bp human FKBP19 mRNA (SEQ ID
NO: 2) sequence is derived from 6 exons on chromosome 12.
TABLE-US-00003 (SEQ ID NO: 2) gaacgagggt cctagctgcc gccacccgaa
cagcctgtcc tggtgccccg gctccctgcc ccgcgcccag tcatgaccct gcgcccctca
ctcctcccgc tccatctgct gctgctgctg ctgctcagtg cggcggtgtg ccgggctgag
gctgggctcg aaaccgaaag tcccgtccgg accctccaag tggagaccct gtggagccc
ccagaaccat gtgccgagcc cgctgctttt ggagacacgc ttcacataca ctacacggga
agcttggtag atggacgtat tattgacacc tccctgacca gagaccctct ggttatagaa
cttggccaaa agcaggtgat tccaggtctg gagcagagtc ttctcgacat gtgtgtggga
gagaagcgaa gggcaatcat tccttctcac ttggcctatg gaaaacgggg atttccacca
tctgtcccag cggatgcagt ggtgcagtat gacgtggagc tgattgcact aatccgagcc
aactactggc taaagctggt gaagggcatt ttgcctctgg tagggatggc catggtgcca
gccctcctgg gcctcattgg gtatcaccta tacagaaagg ccaatagacc caaagtctcc
aaaaagaagc tcaaggaaga gaaacgaaac aagagcaaaa agaaataata aataataaat
tttaaaaaac ttaaaaaaaa aaaaaaaaaa aaaaaaa.
[0079] In some embodiments, nucleic acids are expressed in cells to
produce recombinant FKBP19. In some embodiments the nucleic acid
molecules themselves are used in the composition. The compositions
can be used in ex vivo and in vivo methods of gene therapy to
increase expression of an active form of an FKBP11 polypeptide, a
variant or a fragment thereof.
[0080] An isolated nucleic acid can be, for example, a DNA
molecule, provided one of the nucleic acid sequences normally found
immediately flanking that DNA molecule in a naturally-occurring
genome is removed or absent. Thus, an isolated nucleic acid
includes, without limitation, a DNA molecule that exists as a
separate molecule independent of other sequences (e.g., a
chemically synthesized nucleic acid, or a cDNA or genomic DNA
fragment produced by PCR or restriction endonuclease treatment), as
well as recombinant DNA that is incorporated into a vector, an
autonomously replicating plasmid, a virus (e.g., a retrovirus,
lentivirus, adenovirus, or herpes virus), or into the genomic DNA
of a prokaryote or eukaryote. In addition, an isolated nucleic acid
can include an engineered nucleic acid such as a recombinant DNA
molecule that is part of a hybrid or fusion nucleic acid. A nucleic
acid existing among hundreds to millions of other nucleic acids
within, for example, a cDNA library or a genomic library, or a gel
slice containing a genomic DNA restriction digest, is not to be
considered an isolated nucleic acid.
[0081] Nucleic acids encoding active FKBP11 peptides may be
optimized for expression in a host. Codons may be substituted with
alternative codons encoding the same amino acid to account for
differences in codon usage between the organism from which the
FKBP11 nucleic acid sequence is derived and the expression host. In
this manner, the nucleic acids may be synthesized using expression
host-preferred codons. Nucleic acids can be in sense or antisense
orientation, or can be complementary to a reference sequence
encoding an FKBP11 peptide. Nucleic acids can be DNA, RNA, or
nucleic acid analogs. Nucleic acid analogs can be modified at the
base moiety, sugar moiety, or phosphate backbone. Such modification
can improve, for example, stability, hybridization, or solubility
of the nucleic acid. Modifications at the base moiety can include
deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine or
5-bromo-2'-deoxycytidine for deoxycytidine. Modifications of the
sugar moiety can include modification of the 2' hydroxyl of the
ribose sugar to form 2'-O-methyl or 2'-O-allyl sugars. The
deoxyribose phosphate backbone can be modified to produce
morpholino nucleic acids, in which each base moiety is linked to a
six membered, morpholino ring, or peptide nucleic acids, in which
the deoxyphosphate backbone is replaced by a pseudopeptide backbone
and the four bases are retained. See, for example, Summerton and
Weller (1997) Antisense Nucleic Acid Drug Dev. 7:187-195; and Hyrup
et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the
deoxyphosphate backbone can be replaced with, for example, a
phosphorothioate or phosphorodithioate backbone, a
phosphoroamidite, or an alkyl phosphotriester backbone.
[0082] Nucleic acids, encoding FKBP11 peptides can be inserted into
vectors for expression in a host cell. In some embodiments the host
cell is a mammalian cell. In other embodiments, the host can be a
prokaryotic cell. The vectors can be used for production of
recombinant protein, or in methods of gene therapy. Host cells
(e.g., a prokaryotic cell or a eukaryotic cell such as a CHO cell)
can be used to, for example, produce the FKBP11peptides described
herein. In some embodiments for in vivo transplantation, the host
cell is preferably a pancreatic cell or progenitor cell, for
example, islet/.beta.-cells of the pancreas.
[0083] Nucleic acids in vectors can be operably linked to one or
more expression control sequences. For example, the control
sequence can be incorporated into a genetic construct so that
expression control sequences effectively control expression of a
coding sequence of interest. Examples of expression control
sequences include promoters, enhancers, and transcription
terminating regions. A promoter is an expression control sequence
composed of a region of a DNA molecule, typically within 100
nucleotides upstream of the point at which transcription starts
(generally near the initiation site for RNA polymerase II). To
bring a coding sequence under the control of a promoter, it is
necessary to position the translation initiation site of the
translational reading frame of the polypeptide between one and
about fifty nucleotides downstream of the promoter. Enhancers
provide expression specificity in terms of time, location, and
level. Unlike promoters, enhancers can function when located at
various distances from the transcription site. An enhancer also can
be located downstream from the transcription initiation site. A
coding sequence is "operably linked" and "under the control" of
expression control sequences in a cell when RNA polymerase is able
to transcribe the coding sequence into mRNA, which then can be
translated into the protein encoded by the coding sequence.
[0084] Methods of making vectors for introduction into a cell of
choice are known in the art. Suitable expression vectors include,
without limitation, plasmids and viral vectors derived from, for
example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes
viruses, cytomegalo virus, retroviruses, vaccinia viruses,
adenoviruses, and adeno-associated viruses. Numerous vectors and
expression systems are commercially available from such
corporations as Novagen (Madison, Wis.), Clontech (Palo Alto,
Calif.), Stratagene (La Jolla, Calif.), and Invitrogen Life
Technologies (Carlsbad, Calif.).
[0085] Vectors can be created using molecular cloning and Gateway
technology (Life Technologies) according to manufacturers protocol.
Vectors contain a promoter derived from cytomegalo virus (CMV).
Crude adenovirus are produced by us using ViraPower Adenoviral
Gateway Expression Kit (Life Technologies) according to
manufacturer's protocol. Crude adenovirus produced by using this
method, can be amplified and purified (to obtain pure/clean and
highly concentrated adenovirus suitable for injection into mice) by
Vector Biolabs (Philadelphia). Mice were injected intravenously
(iv) with adenovirus diluted in sterile saline via the tail
vein.
[0086] Callejas, et al., describe treatment of diabetic dogs by
gene therapy, using a one-time intramuscular administration of
adeno-associated viral vector. Callehas, Diabetes, Feb. 1, 2013,
epub ahead of print. Other studies showing successful use of
vectors to delivery genes in humans include Morgan, et al.,
Science, 314(5796):126-9 (2006) (describe conferring tumor
recognition by autologous lymphocytes from peripheral blood by
using a retrovirus that encodes a T cell receptor); Levine, et al,
Proc. natl. Acad. Sci., 103(46):17372-7 (2006) describe lentiviral
vectors that can be used for gene transfer to humans.
[0087] Vectors containing nucleic acids to be expressed can be
transferred into host cells. Although not limited to a particular
technique, a number of these techniques are well established within
the art. In some embodiments for in vivo transplantation, the host
cell is preferably a pancreatic cell or progenitor cell, for
example, islet cells/.beta.-cells of the pancreas. Methods for
isolating host cells, for example, islet cells, are known in the
art and are described for example in U.S. Publication No.
2009/0191608. Methods for in vitro transfection and in vivo
transfer of islet cells to a subject, as well as methods for
protecting in vivo islet grafts are known in art. (Reviewed in
Ajit, et al. Pharmacological reviews, 58(2):194-243 (2006). See
also, U.S. Published Application Nos. 2005/0048040, 2011/0008343
and 2011/0182979.
[0088] C. Compounds Modifying FKBP11 Activity
[0089] FKBP11 is involved in maintaining glucose homeostasis in
obese and type 2 diabetic mice, as well as in a mouse model of type
1 diabetes. FKBP11 expression is dynamically regulated in healthy
lean mice that are subjected to metabolic stress such as refeeding
after a fasting period, indicating an important physiological role
in metabolic control. Hepatic expression levels of FKBP11 are
reduced in obese and type 2 mice. The examples show that restoring
FKBP11 levels dramatically reduced fed and fasted blood glucose
levels, and improved glucose tolerance, hepatic gluconeogenic
activity and insulin sensitivity. FKBP11 expression also reduced
glucose levels in a mouse model of t e-I diabetes.
[0090] Accordingly, compounds which increase FKBP11 levels or
activity or otherwise decrease ER stress through this pathway can
be used to maintain or enhance glucose homeostasis, glucose
tolerance, hepatic gluconeogenic activity and decrease insulin
sensitivity.
[0091] Compounds which may be useful in elevating FKBP11 activity
or levels and thereby improving glucose homeostasis, may be
identified using a variety of known methods, including the animal
models described in the examples.
[0092] D. Dosage Forms
[0093] Pharmaceutical compositions containing the FKBP11 peptides
may be administered parenterally to subjects in need of such a
treatment. Parenteral administration can be performed by
subcutaneous, intramuscular or intravenous injection by means of a
syringe, optionally, a pen-like syringe. Alternatively, parenteral
administration can be performed by means of an infusion pump.
Alternatively, the peptides are administered orally, nasally or
pulmonally, preferably in compositions, powders or liquids,
specifically designed for the purpose.
[0094] The peptides or nucleic acids described herein can be
formulated for parenteral administration. Parenteral formulations
can be prepared as aqueous compositions using techniques is known
in the art. Typically, such compositions are prepared as injectable
formulations, for example, solutions or suspensions; solid forms
suitable for using to prepare solutions or suspensions upon the
addition of a reconstitution medium prior to injection; emulsions,
such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions,
and microemulsions thereof, liposomes, or emulsomes. The carrier
can be a solvent or dispersion medium containing, for example,
water, ethanol, one or more polyols (e.g., glycerol, propylene
glycol, and liquid polyethylene glycol), oils, such as vegetable
oils (e.g., peanut oil, corn oil, sesame oil, etc.), and
combinations thereof.
[0095] The parenteral formulations can be formulated for controlled
release including immediate release, delayed release, extended
release, pulsatile release, and combinations thereof. For example,
the compounds and/or one or more additional active agents can be
incorporated into polymeric microparticles which provide controlled
release of the drug(s). Release of the drug(s) is controlled by
diffusion of the drug(s) out of the microparticles and/or
degradation of the polymeric particles by hydrolysis and/or
enzymatic degradation. Suitable polymers include ethylcellulose and
other natural or synthetic cellulose derivatives. Polymers which
are slowly soluble and form a gel in an aqueous environment, such
as hydroxypropyl methylcellulose or polyethylene oxide may also be
suitable as materials for drug containing microparticles. Other
polymers include, but are not limited to, polyanhydrides,
poly(ester anhydrides), polyhydroxy acids, such as polylactide
(PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA),
poly-3-hydroxybutyrate (PHB) and copolymers thereof,
poly-4-hydroxybutyrate (P4HB) and copolymers thereof,
polycaprolactone and copolymers thereof, and combinations
thereof.
[0096] Pharmaceutical compositions may be formulated in a
conventional manner using one or more physiologically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Formulation of drugs is discussed in, for
example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa. (1975), and Liberman, H. A. and
Lachman, L , Eds., Pharmaceutical Dosage Forms, Marcel Decker, New
York, N.Y. (1980). Proper formulation is dependent upon the route
of administration chosen.
[0097] Pharmaceutically acceptable excipients that can be present
in the FKBP11peptide-containing dosage forms include, but are not
limited to, diluents, binders, lubricants, disintegrants,
colorants, stabilizers, and surfactants. If desired, the tablets,
wafers, films, lozenges, beads, granules, or particles may also
contain minor amount of nontoxic auxiliary substances such as dyes,
sweeteners, coloring and flavoring agents, pH buffering agents, or
preservatives.
[0098] Solutions and dispersions of the active compounds as the
free acid or base or pharmacologically acceptable salts thereof can
be prepared in water or another solvent or dispersing medium
suitably mixed with one or more pharmaceutically acceptable
excipients including, but not limited to, surfactants, dispersants,
emulsifiers, pH modifying agents, and combination thereof. In one
embodiment, a subcutaneous injectable formulation is produced by
mixing an FKBP11 peptide with saline to form a solution and
sterilizing the solution (referred to as the "diluent"). The FKBP11
peptide is separately added to sterile water to form a solution,
filtered, and a designated amount is placed into each of a number
of separate sterile injection bottles. The FKBP11 peptide solution
may be lyophilized to form a powder which can be stored separately
from the diluent to retain its stability. Prior to administration,
the diluent is added to the FKBP11 peptide injection bottle.
[0099] The formulation is typically buffered to a pH of 3-8 for
parenteral administration upon reconstitution. Suitable buffers
include, but are not limited to, phosphate buffers, acetate
buffers, and citrate buffers
[0100] Water soluble polymers are often used in formulations for
parenteral administration. Suitable water-soluble polymers include,
but are not limited to, polyvinylpyrrolidone, dextran,
carboxymethylcellulose, and polyethylene glycol.
[0101] Alternatively, the FKBP 11 peptides can be incorporated into
microparticles prepared from materials which are insoluble in
aqueous solution or slowly soluble in aqueous solution, but are
capable of degrading within the GI tract by means including
enzymatic degradation, surfactant action of bile acids, and/or
mechanical erosion. As used herein, the term "slowly soluble in
water" refers to materials that are not dissolved in water within a
period of 30 minutes. Preferred examples include fats, fatty
substances, waxes, wax-like substances and mixtures thereof.
Suitable fats and fatty substances include fatty alcohols (such as
lauryl, myristyl stearyl, cetyl or cetostearyl alcohol), fatty
acids and derivatives, including, but not limited to, fatty acid
esters, fatty acid glycerides (mono-, di- and tri-glycerides), and
hydrogenated fats. Specific examples include, but are not limited
to hydrogenated vegetable oil, hydrogenated cottonseed oil,
hydrogenated castor oil, hydrogenated oils available under the
trade name Sterotex.RTM., stearic acid, cocoa butter, and stearyl
alcohol. Suitable waxes and wax-like materials include natural or
synthetic waxes, hydrocarbons, and normal waxes. Specific examples
of waxes include beeswax, glycowax, castor wax, carnauba wax,
paraffins and candelilla wax. As used herein, a wax-like material
is defined as any material which is normally solid at room
temperature and has a melting point of from about 30 to 300.degree.
C.
III. Kits
[0102] The FKBP 11 peptide or a fusion protein containing the
FKBP11-peptide can be provided in a kit for use in treating a
subject with diabetes. Kits can include one or more containers
containing a pharmaceutical composition including a therapeutically
effective amount of a specific activator of an FKBP 11 polypeptide,
a variant or a fragment therof. Such kits can further include, if
desired, one or more of various conventional pharmaceutical kit
components, such as, for example, containers with one or more
pharmaceutically acceptable carriers as will be readily apparent to
those skilled in the art. The kit may also include means of
administration, such as one or more of a syringe (e.g., a barrel
syringe or a bulb syringe), intravenous (IV) bag, IV line, IV
needle, and/or cannula. Printed instructions, either as inserts or
as labels, indicating quantities of the components to be
administered, guidelines for administration, and/or guidelines for
mixing the components, can also be included in the kit.
[0103] The FKBP 11 peptide can be stored in one container and the
excipients can be stored in a second container Immediately prior to
administration the contents of both containers are mixed.
[0104] In one embodiment, the kit may contain a vial containing
powdered peptide in the cap, separated by a seal which can be
broken by rotation of the cap, to allow the insulin to mix with the
excipient solution in the vial.
IV. Methods of Using the Compositions
[0105] The compositions described herein are administered to a
subject to lower blood glucose levels, to improve glucose
tolerance, decrease hepatic gluconeogenic activity and/or insulin
sensitivity in the subject. The subject is preferably a mammal,
more preferably, a human subject. Representative subjects include
type 1 diabetics, type II diabetics, obese subjects, subjects
exhibiting higher than normal blood glucose levels, and gestational
diabetics.
[0106] Normal fasting glucose levels are generally less than about
110 mg/dL. Shortly after eating, the blood glucose level may rise
temporarily up to 140 mg/dL. Fasting blood glucose levels over 126
mg/dL, and plasma glucose 2 hours after eating over 200 mg/dL, are
indicative of metabolic disorders, such as type-2 diabetes.
Therefore, in preferred embodiments, the pharmaceutical
compositions are administered in amounts effective to reduce
fasting blood glucose levels in the subject to less than 130 mg/dL,
preferably less than 110 mg/dL, and/or the plasma glucose 2 hours
after eating to less than 200 mg/dL, preferably less than 140
mg/dL.
[0107] Efficacy of the disclosed methods can be monitored by
measuring changes in blood glucose levels, glucose tolerance,
hepatic gluconeogenic, and/or insulin sensitivity content. A
statistically significant change in any of these parameters can be
considered evidence of therapeutic efficacy. It is preferred that a
given marker change by at least 5%, at least 10%, at least 20%, at
least 30%, at least 50% or more in effective therapy. Dosage of the
pharmaceutical compositions can be modified by the physician to
increase efficacy while avoiding side effects or toxicity.
[0108] The formulations containing an FKBP11 peptide, nucleic acid
molecules encoding the FKBP11 peptide, or compound increasing the
activity or levels of an FKBP11 peptide, will be administered in an
appropriate vehicle and route for the compound to be delivered, for
example, via injection (intravenous, intramuscular,
intraperitoneally), topically to a mucosal surface (ocularly,
pulmonary, nasal, buccal, rectal or sublingual), or orally.
[0109] Nucleic acids encoding an FKBP11 peptide can be administered
to subjects in need thereof. Nucleic delivery involves introduction
of "foreign" nucleic acids into a cell and ultimately, into a live
animal. In vivo methods permit direct introduction of the gene
therapy agent into the body. Ex vivo methods are where certain
cells are removed from a human, the gene therapy agent introduced
and the cells returned into the body. Methods which are well known
to those skilled in the art may be used to construct expression
vectors containing sequences encoding polypeptides of interest and
appropriate transcriptional and translational control elements.
These methods include in vitro recombinant DNA techniques,
synthetic techniques, and in vivo genetic recombination. Such
techniques are described in Sambrook et al., Molecular Cloning, A
Laboratory Manual (Cold Spring Harbor Press, 4.sup.th ed.
Plainview, N.Y., 2012)).
[0110] Compositions and methods for delivering nucleic acids to a
subject or cell are known in the art (see U.S. Publication Nos.
2014/0065204, 2014/0073053; U.S. Pat. No. 7,807618; Li, et al.,
Pharm Res., 24(3:438-49 (2007); Grigsby, et al., Scientific
Reports, 2013 Nov. 6; 3:3155. doi: 10.1038/srep03155.
[0111] One approach of delivering the nucleic acids disclosed
herein includes nucleic acid transfer into primary cells in culture
followed by autologous transplantation of the ex vivo transformed
cells into the host, either systemically or into a particular organ
or tissue. Ex vivo methods can include, for example, the steps of
harvesting cells from a subject, culturing the cells, transducing
them with an expression vector, and maintaining the cells under
conditions suitable for expression of the encoded FKBP11 peptide.
These methods are known in the art of molecular biology. The
transduction step can be accomplished by any standard means used
for ex vivo gene therapy, including, for example, calcium
phosphate, lipofection, electroporation, viral infection, and
biolistic gene transfer.
[0112] Alternatively, liposomes or micro- and nanoparticles and
polycations such as asialoglycoprotein/polylysine can be used.
Cells that have been successfully transduced can be selected, for
example, for expression of the coding sequence or of a drug
resistance gene. The cells then can be lethally irradiated (if
desired) and injected or implanted into the subject.
[0113] In vivo nucleic acid therapy can be accomplished by direct
transfer of a functionally active DNA into mammalian somatic tissue
or organ in vivo. Nucleic acids may also be administered in vivo by
viral means. Nucleic acid molecules encoding an FKBP11 peptide may
be packaged into retrovirus vectors using packaging cell lines that
produce replication-defective retroviruses, as is well-known in the
art. Other virus vectors may also be used, including recombinant
adenoviruses and vaccinia virus, which can be rendered
non-replicating. In addition to naked DNA or RNA, or viral vectors,
engineered bacteria may be used as vectors.
[0114] The FKBP11 peptide may be administered alone, or in
combination with other bioactive agents. Suitable bioactive agents
include diabetes medications, which include insulin and insulin
analogs, sulfonylureas, meglitinides, biguanides,
thiazolidinediones, alpha-glucosidase inhibitors, or DPP-4
inhibitors. Sulfonylureas stimulate the beta cells of the pancreas
to release more insulin. Chlorpropamide (Diabinese) is the only
first-generation sulfonylurea still in use today. The second
generation sulfonylureas are used in smaller doses than the
first-generation drugs. There are three second-generation drugs:
glipizide (Glucotrol and Glucotrol XL), glyburide (Micronase,
Glynase, and Diabeta), and glimepiride (Amaryl). Meglitinides are
drugs that also stimulate the beta cells to release insulin.
Repaglinide (Prandin) and nateglinide (Starlix) are meglitinides.
Metformin (Glucophage) is a biguanide. Biguanides lower blood
glucose levels primarily by decreasing the amount of glucose
produced by the liver. Rosiglitazone (Avandia) and pioglitazone
(ACTOS) are in a group of drugs called thiazolidinediones. These
drugs help insulin work better in the muscle and fat and also
reduce glucose production in the liver. DPP-4 inhibitors help
improve A1C without causing hypoglycemia. They work by preventing
the breakdown of a naturally occurring compound in the body, GLP-1.
GLP-1 reduces blood glucose levels in the body, but is broken down
very quickly so it does not work well when injected as a drug
itself By interfering in the process that breaks down GLP-1, DPP-4
inhibitors allow it to remain active in the body longer, lowering
blood glucose levels only when they are elevated. Sitagliptin
(JANUVIA) and saxagliptin (ONGLYZA) are the two DPP-4 inhibitors
currently on the market.
[0115] Screening Assays
[0116] In general, candidate agents can be identified from large
libraries of natural products or synthetic (or semi-synthetic)
extracts or chemical libraries according to methods known in the
art. Those skilled in the field of drug discovery and development
will understand that the precise source of test extracts or
compounds is not critical to the screening procedure(s).
[0117] Virtually any number of chemical extracts or compounds can
be screened using the exemplary methods described herein. Examples
of such extracts or compounds include, but are not limited to,
plant-based, fungal-based, prokaryotic-based, or animal-based
extracts, fermentation broths, and synthetic compounds, as well as
modification of existing compounds. Numerous methods are also
available for generating random or directed synthesis (e.g.,
semi-synthesis or total synthesis) of any number of chemical
compounds, including, but not limited to, saccharide-based,
lipid-based, peptide-based, polypeptide-based and nucleic
acid-based compounds. Synthetic compound libraries and libraries of
natural compounds in the form of bacterial, fungal, plant, and
animal extracts are commercially available from a number of
sources. In addition, natural and synthetically libraries can be
produced, if desired, according to routine methods, e.g., by
standard extraction and fractionation methods. Furthermore, if
desired, any library or compound is readily modified using standard
chemical, physical, or biochemical methods.
[0118] When a crude extract is found to have a desired activity,
further fractionation of the positive lead extract may be necessary
to isolate chemical constituents responsible for the observed
effect. The goal of the extraction, fractionation, and purification
process is the careful characterization and identification of a
chemical entity within the crude extract having the desired
activity. Assays can be used to purify the active component and to
test derivatives thereof. Methods of fractionation and purification
of such heterogenous extracts are known in the art. If desired,
compounds shown to be useful agents for treatment are chemically
modified according to methods known in the art. Compounds
identified as being of therapeutic value may be subsequently
analyzed using appropriate in vitro or animal models, for example,
animal models of type 1 and/or type 2 diabetes.
[0119] Candidate agents encompass numerous chemical classes, but
are most often organic molecules, e.g., small organic compounds
having a molecular weight of more than 100 and less than about
2,500 daltons. Candidate agents contain functional groups necessary
for structural interaction with proteins, particularly hydrogen
bonding, and typically include at least an amine, carbonyl,
hydroxyl or carboxyl group, for example, at least two of the
functional chemical groups. The candidate agents often contain
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0120] For example, a microarray analysis on livers of mice that
overexpress FKBP11 can be performed, providing information on
FKBP11-mediated changes in gene expression in the liver. A similar
methodology in an in vitro setting in which cells (preferably
mammalian) are treated with candidate agents can be used to
identify agents that induce similar gene expression patterns like
FKBP11. Candidate agents that induce changes in gene expression
that are similar to changes mediated by FKBP11, those candidates
could be further tested for their potential effect on FKBP11
action/activity. In addition, potential candidates can be tested
for their effect on glucose and insulin metabolism in in vitro or
in vivo settings.
[0121] Cheminformatics and in-silico predictive models are used to
increase the efficiency of the experimental approaches. Additional
information such as compound-target interactions, target-mechanism
of action/pathway relationships, and target-disease associations
can be mined from internal and publically available external
databases. The combination of experimental and predicted
compound-target pharmacological profiles can be used to prioritize
compounds for additional screening and to provide evidence for
proposed mechanisms of action. In addition, these profiles can be
used to retrieve similar compounds for additional testing.
[0122] Chemogenomics library represents an additional opportunity
to identify a biological target. To address the limitation of a
suitable screening collection for use in phenotypic assays, the
Chemogenomics screening collection was constructed in 2011.
Chemogenomics sets consist of .about.5,000 compounds covering
>1,000 targets. Compounds screening set is created based on
single targets or clustered biology space. These compound sets
(10-20 compounds) provide an additional set of tools to confirm the
biology space identified by their Chemogenomics screening hits.
[0123] Chemicals identified in addition to those already known to
target the pathway should lead to additional compounds related in
the targets or activity of the known compounds and these can be
identified by the informatics tools. A significant portion of the
screen will be a pathway enriched screen. Screening with compounds
of known biological mechanism-of-action reduces transition time
from the primary stage to a more focused screen based on improved
selectivity and chemical properties.
[0124] Two strategies can be employed for compound selection. The
first strategy is based on the identification of alternative
targets from the bioinformatics screening to be performed.
Compounds can be selected based on their selectivity profile, as
well as chemical properties. The second strategy will select
compounds following screening of compounds from focused chemical
libraries, such as the chemogenomics set. This provides a library
of up to 5000 compounds that covers .about.1000 biological targets
for a full phenotypic screen. In combination with the
bioinformatics results, appropriate compounds will be used for
screening in the mouse model.
[0125] The use of chemoinfoimatics and in silico models can be
employed to examine data from various studies. All of the compound
efficacy data from screenings is mapped to targets and those
targets used for a pathway-enrichment analysis. Component genes
from pathways containing a significantly enriched number of
screening hits can then be used to query the drug library.
Compounds that target genes from the expanded pathways will then be
selected for follow-up analysis in the animal models. The
combination of experimental and predicted compound-target
pharmacological profiles can be used to prioritize compounds for
additional screening and to provide evidence for proposed
mechanisms of action. In addition, these profiles can be used to
retrieve similar compounds for additional testing.
[0126] The host cells described therein can be employed in a
screening assay, to identify agents which upregulate/inhibit FKBP
11 activity within the context of glucose metabolism.
[0127] The present invention will be further understood by
reference to the following non-limiting examples.
EXAMPLES
Example 1
Expression Levels of FKBP11 in Obese (ob/ob) and Lean Mice
[0128] Materials and Methods
[0129] Livers were obtained from 6 hr fasted lean and ob/ob mice.
Livers were rapidly snap-frozen in liquid nitrogen and stored at
-80 C until further processing. For protein isolation, small pieces
of liver (-100mg) were homogenized in tissue lysis buffer. FKBP11
protein expression in liver lysates was determined using western
blot analysis. For RNA isolation, small pieces (.about.50 mg) of
liver were homogenized in QIAzol reagent (Qiagen). RNA was isolated
using chloroform extraction and subsequent isopropanol
precipitation. cDNA was produced using iScript cDNA synthesis kit
(Biorad). Gene expression was analyzed by QPCR using SYBR green
reagent and iCycler instrument. Relative gene expression levels
were determined using delta Ct method.
[0130] Results
[0131] FIGS. 1A-1B show that hepatic gene expression levels of
FKBP11 are reduced in obese and type 2 diabetic mice and in a high
fat diet (HFD)-induced obese and insulin resistant mice when
compared to lean mice. A similar pattern was seen with protein
expression levels.
Example 2
Effect of Restored FKBP11 Expression on Glucose Tolerance, Hepatic
Gluconeogenic Activity and Insulin Sensitivity
[0132] Materials and Methods
[0133] Mice were intravenously injected with control (adLacZ) or
FKBP11-containing adenovirus via the tail vein. Body weight, food
intake and blood glucose levels were measured every other day. Five
days after injection, mice were subjected to a glucose tolerance
test (GTT). Mice were fasted overnight. In the morning, mice were
intraperitoneally injected with a bolus of glucose and blood
glucose concentrations were measured in time using a Contour
glucose meter (Bayer). Seven days after adenovirus injection, mice
were subjected to an insulin tolerance test (ITT). Mice were fasted
for 6 hrs and subsequently intraperitoneally injected with a bolus
of insulin. Blood glucose concentrations were measured in time
using a Contour glucose meter (Bayer). Mice were killed after a 6
hrs fast on day nine after adenovirus injection.
[0134] Results
[0135] Overexpression of FKBP11 in livers of lean mice does not
affect body weight (FIG. 2B), food intake (FIG. 2C) and blood
glucose levels (FIG. 2D). Similarly, overexpression of FKBP11 in
livers of ob/ob mice does not affect body weight (FIG. 3C) or food
intake (FIG. 3D), but it significantly lowers blood glucose levels
(FIG. 3E) on ob/ob mice. By contrast, overexpression of FKBP11 in
livers of lean mice does not affect body weight (FIG. 2B), food
intake (FIG. 2C) and blood glucose levels (FIG. 2C).
Example 3
The Effect of FKBP11 Overexpression in an In Vivo Model of Type 1
Diabetes
[0136] Materials and Methods
[0137] Type I diabetes was induced by injecting C57B6/J mice with
streptozotocin (STZ). Diabetes, as determined by glucose levels
>500 mg/dl develops within 4 days. Mice that did not meet these
criteria were not included in the study. Type 1 diabetic mice were
intravenously injected with control (adLacZ) or FKBP11-containing
adenovirus via the tail vein. Body weight, food intake and blood
glucose levels were measured every other day.
[0138] Results
[0139] FKBP11 overexpression in HDF-fed and STZ-induced type 1
diabetic mice does not affect body weight (FIGS. 6C and 7F) or food
intake (FIGS. 6B and 7E), but it significantly lowers blood glucose
levels (FIGS. 6E and 7F). Glucose tolerance (as assessed by glucose
tolerance test GTT), hepatic gluconeogenic activity (as assessed by
pyruvate tolerance test, PTT) and insulin sensitivity (as assessed
by insulin tolerance, ITT), in ob/ob mice overexpressing FKBP11 are
dramatically improved compared to mice that expressed a control
virus (FIGS. 4D-4F and 5C-5D). FKBP11 overexpression in HDF-fed
mice significantly improves glucose tolerance as assessed by GTT
(FIGS. 6E and F). By contrast, FKBP11 overexpression does not
improve glucose tolerance or insulin sensitivity in lean mice (FIG.
4A-4C) but it improves hepatic gluconeogenic activity (FIGS. 5A and
5B).
[0140] FKBP11 overexpression does not affect insulin levels (FIG.
7C), body weight (FIG. 7D) or food intake (FIG. 7E) in
streptozotocin (STZ)-induced type 1 diabetic mice.
Example 4
Secretion of FKBP11 in Lean and Obese Mice/MICE Overexpressing
FKBP11
[0141] Materials and Methods
[0142] Lean and ob/ob mice were killed by cardiac puncture under
isoflurane anesthesia after a 6 hr fast. Mice overexpressing LacZ
or FKBP11 (adenovirus-mediated overexpression, intravenously
injected via the tail vein) were killed by cardiac puncture under
isoflurane anesthesia after a 6 hr fast on day 4 after adenovirus
injection. Blood was collected in heparin-coated tubes and
centrifuged at 4 degrees to obtain the plasma. Plasma was cleaned
from albumin/IgG and loaded onto SDS gels. FKBP11 was visualized
using western blot analysis. An ELISA was developed. Plates were
coated with an FKBP11 antibody (raised in goat) and subsequently
incubated with media from cells that overexpressed FKBP11. Bound
FKBP11 was detected using a second FKBP11 antibody (raised in
rabbit) and visualized using HRP labeled antibody and Turbo TMB
ELISA reagent.
[0143] Results
[0144] Mice that overexpress FKBP11 in the liver have higher plasma
levels of FKBP11 in as seen in studies using two different cohorts
of mice) (data not shown). The presence of FKBP11 in cell culture
medium was detected following FLAG tagged-FKBP11 expression and
western blot analysis for FLAG in the cell culture media (data not
shown). FKBP11 ELISA read outs (A450 nm) from cell culture media of
HEK cells overexpressing FKBP11 are presented in FIG. 8.
Example 5
A single, Intravenous Injection of Recombinant Full-Length FKBP11
Reduces Fasting Blood Glucose
[0145] Materials and Methods
[0146] To induce obesity, wt mice (C57BL/6J) were fed a high-fat
diet (45 kcal % fat) for six months. After establishment of
obesity, mice were intravenously injected with 10 mg/kg recombinant
FKBP11 (rFKBP11) or corresponding solvent via the tail vein. After
an overnight (10 PM-9 AM) fast, blood glucose levels were measured
using a Contour glucose meter (Bayer)
[0147] The results (FIG. 9) show that a single, intravenous
injection of recombinant full-length FKBP11 reduces fasting blood
glucose in obese and diabetic mice. This is further evidence of the
potential importance of circulating FKBP11 in regulation of glucose
metabolism.
[0148] Discussion
[0149] Sulten, et al. Mamm. Genome, 17(4):322-331 (2006), reviewed
the expression profile of FKBP19, and concluded that it suggests a
unique role for FKBP19 in protein secretion. Other studies have
identified FKBP11 as a potential marker for diagnosis of diabetes.
For example, EP 1840573 lists FKBP11 as an example of a marker
which could be used to diagnose a disease or a predisposition to a
disease having a preinflammatory phase, for example, diabetes,
before any clinical symptom of the disease is apparent. U.S. Pat.
Nos. 7,951,776 and 7,951,382, identify biological markers
associated with the risk of developing diabetes, as well as methods
of using such biological markers in diagnosis and prognosis of
diabetes. FKBP11 is among the five hundred and forty eight (548) of
the markers thus identified. Lu, et al., Mol. Cell. Prot.,
7(8):1434-1450 (2008) describe a study associating 159 proteins
(including FKBP11), with islet dysfunction. Lu, et al., disclose
that FKBP11 and FKBP2, among many other proteins, are highly
upregulated in the islets from a mouse model of insulin
resistance.
[0150] By contrast, the studies described in this application show
a direct link between low levels of secreted FKBP11 and glucose
metabolism. The Examples show that FKBP11 is a crucial player in
maintenance of glucose homeostasis in obese and type 2 diabetic
mice as well as in a mouse model of type 1 diabetes. Hepatic
expression levels of FKBP11 are reduced in obese and type 2
diabetic mice and in a high fat diet (HFD)-induced obese and
insulin resistant mice when compared to lean mice (FIG. 1A-1B).
[0151] An adenoviral-mediated approach to restore FKBP11 expression
in obese mice dramatically reduced both fasted blood glucose levels
in obese mice.
[0152] Overexpression of FKBP11 in livers of ob/ob mice does not
affect body weight (FIG. 3C) or food intake (FIG. 3D), but it
significantly lowers blood glucose levels (FIG. 3E) on ob/ob mice.
The same results were obtained with HDF-fed and STZ-induced type 1
diabetic mice that overexpress FKBP11. FKBP11 overexpression in
HDF-fed and STZ-induced type 1 diabetic mice does not affect body
weight (FIGS. 6C and 7F) or food intake (FIGS. 6B and 7E), but it
significantly lowers blood glucose levels (FIGS. 6D and 7F). By
contrast, overexpression of FKBP11 in livers of lean mice does not
affect body weight (FIG. 2C), food intake (FIG. 2D) and blood
glucose levels (FIG. 2E).
[0153] In addition, glucose tolerance (as assessed by glucose
tolerance test GTT), hepatic gluconeogenic activity and insulin
sensitivity in ob/ob mice overexpressing FKBP11 are dramatically
improved compared to mice that expressed a control virus (FIGS.
4D-4F and 5C-5D). FKBP11 overexpression in HDF-fed mice
significantly improves glucose tolerance as assessed by GTT (FIGS.
6F and F). By contrast, FKBP11 overexpression does not improve
glucose tolerance or insulin sensitivity in lean mice (FIG. 4A-4C)
but it improves hepatic gluconeogenic activity (FIGS. 5A and
5B).
[0154] FKBP11 overexpression does not affect insulin levels (FIG.
7E), body weight (FIG. 7D) or food intake (FIG. 7E) in
streptozotocin (STZ)-induced type 1 diabetic mice.
[0155] Expression of FKBP11 at high levels is not required for the
effects described here. Rather, restoring FKBP11 expression levels
to levels observed in lean healthy controls is sufficient to
recover glucose tolerance and insulin sensitivity in obese
mice.
[0156] These results confirm the biological significance of FKBP11
in regulation of glucose homeostasis, and provide important
therapeutic potential for treatment of hyperglycemia in both type 1
and type 2 diabetes.
[0157] FKBP11 is predicted to reside in the ER membrane as a type 1
transmembrane protein. In addition to its broad tissue expression
pattern, FKBP11 has detected in the circulation of mice.
Significant levels of FKBP11 were detected in the circulation of
mice. Further, FKBP11 overexpressed in livers of lean mice was
subsequently detected at significantly increased levels in the
plasma of these mice (data not shown). FKBP11 is potentially
cleaved in a yet unknown manner followed by secretion into the
circulation where it might function as a hormone. Corresponding
with the observation that obese mice have reduced hepatic FKBP11,
plasma levels of FKBP11 also appear to be reduced in these mice.
While not been bound by theory, secreted FKBP11 may be functioning
as a hormone, regulating glucose metabolism; this provides numerous
potential possibilities for the development of therapeutic
interventions for the treatment of both type 2 and type 1 diabetes.
Sequence CWU 1
1
101201PRTArtificial SequenceSynthetic Polypeptide 1Met Thr Leu Arg
Pro Ser Leu Leu Pro Leu His Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu
Ser Ala Ala Val Cys Arg Ala Glu Ala Gly Leu Glu Thr Glu 20 25 30
Ser Pro Val Arg Thr Leu Gln Val Glu Thr Leu Val Glu Pro Pro Glu 35
40 45 Pro Cys Ala Glu Pro Ala Ala Phe Gly Asp Thr Leu His Ile His
Tyr 50 55 60 Thr Gly Ser Leu Val Asp Gly Arg Ile Ile Asp Thr Ser
Leu Thr Arg 65 70 75 80 Asp Pro Leu Val Ile Glu Leu Gly Gln Lys Gln
Val Ile Pro Gly Leu 85 90 95 Glu Gln Ser Leu Leu Asp Met Cys Val
Gly Glu Lys Arg Arg Ala Ile 100 105 110 Ile Pro Ser His Leu Ala Tyr
Gly Lys Arg Gly Phe Pro Pro Ser Val 115 120 125 Pro Ala Asp Ala Val
Val Gln Tyr Asp Val Glu Leu Ile Ala Leu Ile 130 135 140 Arg Ala Asn
Tyr Trp Leu Lys Leu Val Lys Gly Ile Leu Pro Leu Val 145 150 155 160
Gly Met Ala Met Val Pro Ala Leu Leu Gly Leu Ile Gly Tyr His Leu 165
170 175 Tyr Arg Lys Ala Asn Arg Pro Lys Val Ser Lys Lys Lys Leu Lys
Glu 180 185 190 Glu Lys Arg Asn Lys Ser Lys Lys Lys 195 200
2726DNAHomo sapiens 2gaacgagggt cctagctgcc gccacccgaa cagcctgtcc
tggtgccccg gctccctgcc 60ccgcgcccag tcatgaccct gcgcccctca ctcctcccgc
tccatctgct gctgctgctg 120ctgctcagtg cggcggtgtg ccgggctgag
gctgggctcg aaaccgaaag tcccgtccgg 180accctccaag tggagaccct
gtggagcccc cagaaccatg tgccgagccc gctgcttttg 240gagacacgct
tcacatacac tacacgggaa gcttggtaga tggacgtatt attgacacct
300ccctgaccag agaccctctg gttatagaac ttggccaaaa gcaggtgatt
ccaggtctgg 360agcagagtct tctcgacatg tgtgtgggag agaagcgaag
ggcaatcatt ccttctcact 420tggcctatgg aaaacgggga tttccaccat
ctgtcccagc ggatgcagtg gtgcagtatg 480acgtggagct gattgcacta
atccgagcca actactggct aaagctggtg aagggcattt 540tgcctctggt
agggatggcc atggtgccag ccctcctggg cctcattggg tatcacctat
600acagaaaggc caatagaccc aaagtctcca aaaagaagct caaggaagag
aaacgaaaca 660agagcaaaaa gaaataataa ataataaatt ttaaaaaact
taaaaaaaaa aaaaaaaaaa 720aaaaaa 726311PRTHuman immunodeficiency
virus 3Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10
49PRTHuman immunodeficiency virus 4Arg Lys Lys Arg Arg Gln Arg Arg
Arg 1 5 512PRTArtificial SequenceProtein transduction domain 5Arg
Arg Gln Arg Arg Thr Ser Lys Leu Met Lys Arg 1 5 10 627PRTArtificial
SequenceProtein transduction domain 6Gly Trp Thr Leu Asn Ser Ala
Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10 15 Lys Ala Leu Ala Ala
Leu Ala Lys Lys Ile Leu 20 25 729PRTArtificial SequenceProtein
transduction domain 7Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala Lys
Ala Leu Ala Lys His 1 5 10 15 Leu Ala Lys Ala Leu Ala Lys Ala Leu
Lys Cys Glu Ala 20 25 816PRTArtificial SequenceProtein transduction
domain 8Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys
Lys 1 5 10 15 920PRTArtificial SequenceEndosomal Escape Sequence
9Gly Asp Ile Met Gly Glu Trp Gly Asn Glu Ile Phe Gly Ala Ile Ala 1
5 10 15 Gly Phe Leu Gly 20 1035PRTArtificial SequenceProtein
transduction domain 10Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Gly Glu Gly Asp Ile 1 5 10 15 Met Gly Glu Trp Gly Asn Glu Ile Phe
Gly Ala Ile Ala Gly Phe Leu 20 25 30 Gly Gly Glu 35
* * * * *